**4.3 Breath collection**

In breath analysis, depending on the desired result, is required a knowledge and understanding of respiratory physics. In the individual, normal, resting breathing is about 0.5 L, breathing known as tidal volume. The total volume of the lung is about

6 L, of which 4.8 L is called the vital capacity and the remaining 1.2 liters is called the residual volume and remains in the lungs [77–79].

Exhaled breath is an inhomogeneous gas mixture composed of "dead space" (roughly 150 milliliters) and the gas part represented by the "alveolar" respiration coming from the lungs (about 350 milliliters). Human exhalation contains both gas molecules resulting from the exchange of blood, but also compounds from the atmosphere. In the process of breath gases collection, there are three basic approaches [79, 80]:


For the desired results must be taken into account different factors such as the type of breath collected, single or multiple exhalation, and the technique used for sample analysis.

#### **4.4 Protocols and procedures for breath analysis**

Our measurement procedure to determine the concentrations of gases involves the following basic steps: cleaning the cell, calibration of the cell or measurement of the cell responsivity, and acquiring spectra of ethylene and ammonia [45, 56, 57].

For the measurement and detection of the gases from breath, the laser is kept tuned where ethylene and ammonia exhibit the strongest and most characteristic peaks. The cleaning of the cell must be carried out by successive washing with nitrogen of purity 6,0 (99,9999%) at atmospheric pressure, cleaning performed each time the contents of the cell are changed. An adequate degree of cell clearance is considered to be obtained if the PA signal measured in the nitrogen atmosphere has a rather low level, usually 30 μV. Absorption measurements are performed at room temperature, in the range 20° C - 22°C.

Exhaled breath samples are collected in aluminized bags (750 ml aluminumcoated bags) consisting of: a disposable mouthpiece, a tee-mouthpiece assembly (including a plastic tee and a removable one-way flutter valve), a bag with the role of collecting "dead air" (the first part of expired breath), while the alveolar air in the collection bag and a discard multi-patient collection bags are designed to collect multiple. The breath sample can be kept in multi-patient collection bags for up to 6 hours.

**157**

*Organic Volatile Compounds Used in Type 2 Diabetes DOI: http://dx.doi.org/10.5772/intechopen.94752*

sample) and water vapor [94].

were excluded from the study.

subjects compared to healthy subjects.

measurement, an intense N2 washing cycle of the PA cell is performed. In this way the PA signal measured is due exclusively to the absorption of ammonia or ethylene molecules. Before measurements the PA cell is filled with pure nitrogen and the background signal detected, and we started from a background signal ~25 μV. After collecting the breath sample, the gas sample is transferred from the bag to the PA cell at a controlled flow rate 36 l h−1 (600 standard cubic centimeters per minute (sccm)). Before the PA cell is found a trap filled (with a volume > 100 cm3

with KOH (potassium hydroxide) pellets replaced after each measurement, pellets used to retain interfering gases such as carbon dioxide (~4% in exhaled breath

fied and is proportional to the ethylene and to the ammonia concentrations.

**5. VOCs measurement from the exhaled breath in type 2 diabetes**

Oxidative damage was quantified by measuring breath ethylene and ammonia concentrations using CO2LPAS system. PA detection provides necessary selectivity for analyzing multicomponent mixtures by the use of line-tunable CO2 lasers. Breath samples were collected in special sample bags, aluminized multi-patient collection bags from QuinTron (750 mL aluminum-coated bags). All samples were given between 09:00 and 11:00 a.m. and analyzed using the CO2LPAS system. Measurements of HbA1c and glucose were done using standard procedure. The subjects involved in this study are persons diagnosed with T2DM (n = 16), recruited from the family doctor, age between 42 and 71 years, body mass index BMI = 31.4–35.3, known stable cases of T2DM whose medical therapy had been unaltered over the last 12 months, and a healthy control group (n = 9), age between 29 and 42 years, non-smokers, non-diabetics and body mass index BMI = 19.8–23.4. From T2DM subjects, 7 present hypertension and inflammatory syndrome. No patients were on supplements with antioxidants. Informed consent was obtained from all individuals. The participants with hormonal disorders, benign or malignant disorders, renal failure, central nervous system disorders, and also smokers

As an observation of the results obtained, it can be seen that the average ethylene of T2DM subjects is higher than the average ethylene healthy subjects (see **Figure 3**). The ethylene values in healthy subjects are normal and in the range 10.73 ppb and 57.13 ppb, but at the subjects with T2DM the ethylene concentrations range was between 78 ppb and 444 ppb. The differences in exhaled breath ammonia concentration are presented in **Figure 4**, where the mean values of breath ethylene concentrations in healthy control group and subjects with T2DM are presented. The ammonia values in healthy subjects are normal and in the range 0.832 ppm and 1.76 ppm, but at the subjects with T2DM the ammonia concentration range was between 2.74 ppm and 10.16 ppm. Our measurements showed a significantly increase of ammonia concentrations in the exhaled breath at diabetic

All of the collected samples were analyzed over a period of three months. To remove any residual contaminants, all of these bags were thoroughly cleaned by flushing with nitrogen gas (purity 99.9999%) and subsequently evacuated for breath sample collection. Following the procedure, the breath samples was introduced in the PA cell, the PA cell closed and used for measurements. The measurements were performed on the 10P (14) laser line (where ethylene exhibits a strong absorption with an absorption coefficient of 30.4 cm−1 atm−1) and 9R (30) (where ammonia exhibits a strong absorption with an absorption coefficient of 57 cm−1 atm−1). In this way the signal measured by microphones in PA cell is quanti-

)

Ammonia is a highly adsorbed compound and the ammonia molecules adhere very well to the walls of the PA cell, so that to ensure the quality of each *Organic Volatile Compounds Used in Type 2 Diabetes DOI: http://dx.doi.org/10.5772/intechopen.94752*

*Type 2 Diabetes - From Pathophysiology to Cyber Systems*

where gas cannot be exchanged.

**4.4 Protocols and procedures for breath analysis**

room temperature, in the range 20° C - 22°C.

approaches [79, 80]:

gas, is collected.

sample analysis.

the residual volume and remains in the lungs [77–79].

6 L, of which 4.8 L is called the vital capacity and the remaining 1.2 liters is called

Exhaled breath is an inhomogeneous gas mixture composed of "dead space" (roughly 150 milliliters) and the gas part represented by the "alveolar" respiration coming from the lungs (about 350 milliliters). Human exhalation contains both gas molecules resulting from the exchange of blood, but also compounds from the atmosphere. In the process of breath gases collection, there are three basic

1.Dead-space gas collection (or upper airway collection), which is the volume of air in the conducting airways where gas exchange cannot take place, where no exchange of oxygen or carbon dioxide occurs. There is also an alveolar dead space, which is the air in those alveoli that are ventilated but not perfused,

2.Alveolar collection (pure alveolar gas is collected), refers at the portion of exhaled breath that contain gases that have exchanged with blood. In the lungs there is a great flow of blood and the exhaled breath that contains both gas molecules resulting from the exchange of blood, but also compounds from the atmosphere. More specifically, excluding the dead-space from the analysis are excluded the chemicals that originate from the atmosphere. Most respiratory tests use this type of test because it contains VOCs resulting from endogenous

3.Mixed breath collection means that total breath, dead-space air and alveolar

For the desired results must be taken into account different factors such as the type of breath collected, single or multiple exhalation, and the technique used for

Our measurement procedure to determine the concentrations of gases involves the following basic steps: cleaning the cell, calibration of the cell or measurement of the cell responsivity, and acquiring spectra of ethylene and ammonia [45, 56, 57]. For the measurement and detection of the gases from breath, the laser is kept tuned where ethylene and ammonia exhibit the strongest and most characteristic peaks. The cleaning of the cell must be carried out by successive washing with nitrogen of purity 6,0 (99,9999%) at atmospheric pressure, cleaning performed each time the contents of the cell are changed. An adequate degree of cell clearance is considered to be obtained if the PA signal measured in the nitrogen atmosphere has a rather low level, usually 30 μV. Absorption measurements are performed at

Exhaled breath samples are collected in aluminized bags (750 ml aluminumcoated bags) consisting of: a disposable mouthpiece, a tee-mouthpiece assembly (including a plastic tee and a removable one-way flutter valve), a bag with the role of collecting "dead air" (the first part of expired breath), while the alveolar air in the collection bag and a discard multi-patient collection bags are designed to collect multiple. The breath sample can be kept in multi-patient collection bags for up to

Ammonia is a highly adsorbed compound and the ammonia molecules adhere very well to the walls of the PA cell, so that to ensure the quality of each

activity and the influence of exogenous VOCs is eliminated.

**156**

6 hours.

measurement, an intense N2 washing cycle of the PA cell is performed. In this way the PA signal measured is due exclusively to the absorption of ammonia or ethylene molecules. Before measurements the PA cell is filled with pure nitrogen and the background signal detected, and we started from a background signal ~25 μV.

After collecting the breath sample, the gas sample is transferred from the bag to the PA cell at a controlled flow rate 36 l h−1 (600 standard cubic centimeters per minute (sccm)). Before the PA cell is found a trap filled (with a volume > 100 cm3 ) with KOH (potassium hydroxide) pellets replaced after each measurement, pellets used to retain interfering gases such as carbon dioxide (~4% in exhaled breath sample) and water vapor [94].

All of the collected samples were analyzed over a period of three months. To remove any residual contaminants, all of these bags were thoroughly cleaned by flushing with nitrogen gas (purity 99.9999%) and subsequently evacuated for breath sample collection. Following the procedure, the breath samples was introduced in the PA cell, the PA cell closed and used for measurements. The measurements were performed on the 10P (14) laser line (where ethylene exhibits a strong absorption with an absorption coefficient of 30.4 cm−1 atm−1) and 9R (30) (where ammonia exhibits a strong absorption with an absorption coefficient of 57 cm−1 atm−1). In this way the signal measured by microphones in PA cell is quantified and is proportional to the ethylene and to the ammonia concentrations.

## **5. VOCs measurement from the exhaled breath in type 2 diabetes**

Oxidative damage was quantified by measuring breath ethylene and ammonia concentrations using CO2LPAS system. PA detection provides necessary selectivity for analyzing multicomponent mixtures by the use of line-tunable CO2 lasers. Breath samples were collected in special sample bags, aluminized multi-patient collection bags from QuinTron (750 mL aluminum-coated bags). All samples were given between 09:00 and 11:00 a.m. and analyzed using the CO2LPAS system. Measurements of HbA1c and glucose were done using standard procedure. The subjects involved in this study are persons diagnosed with T2DM (n = 16), recruited from the family doctor, age between 42 and 71 years, body mass index BMI = 31.4–35.3, known stable cases of T2DM whose medical therapy had been unaltered over the last 12 months, and a healthy control group (n = 9), age between 29 and 42 years, non-smokers, non-diabetics and body mass index BMI = 19.8–23.4. From T2DM subjects, 7 present hypertension and inflammatory syndrome. No patients were on supplements with antioxidants. Informed consent was obtained from all individuals. The participants with hormonal disorders, benign or malignant disorders, renal failure, central nervous system disorders, and also smokers were excluded from the study.

As an observation of the results obtained, it can be seen that the average ethylene of T2DM subjects is higher than the average ethylene healthy subjects (see **Figure 3**). The ethylene values in healthy subjects are normal and in the range 10.73 ppb and 57.13 ppb, but at the subjects with T2DM the ethylene concentrations range was between 78 ppb and 444 ppb. The differences in exhaled breath ammonia concentration are presented in **Figure 4**, where the mean values of breath ethylene concentrations in healthy control group and subjects with T2DM are presented. The ammonia values in healthy subjects are normal and in the range 0.832 ppm and 1.76 ppm, but at the subjects with T2DM the ammonia concentration range was between 2.74 ppm and 10.16 ppm. Our measurements showed a significantly increase of ammonia concentrations in the exhaled breath at diabetic subjects compared to healthy subjects.

For the subjects involved in this study, among the exhaled breath analyses, were determined blood analyses such as blood glucose levels and glycated hemoglobin HbA1C. **Table 1** shows the values of HbA1c and glucose blood tests, as well as the values of the respiration results obtained with the CO2LPAS system.

Among subjects with T2DM, 7 have hypertension and inflammatory syndrome. In those subjects averages of all analyzes, both blood tests and the breathing are

**Figure 3.** *Mean breath ethylene concentrations in healthy subjects and T2DM.*

**Figure 4.** *Mean breath ammonia concentrations in healthy subjects and T2DM.*


**159**

tive stress.

**Figure 5.**

**6. Discussions**

*Organic Volatile Compounds Used in Type 2 Diabetes DOI: http://dx.doi.org/10.5772/intechopen.94752*

higher in subjects with T2DM who have hypertension and inflammatory syndrome

Our results show a high difference in the HbA1c mean value of healthy group and the and the mean values obtained in subjects with T2DM. These differences are presented in **Figure 5**. In diabetes, impaired detoxification of free radicals and this degree of damage can be seen in HbA1c, which is a biomarker of oxida-

Damage to proteins and lipids due to oxidation has been implicated in the pathogenesis of type diabetes. Previous studies on oxidative stress are based on invasive blood samples analysis, and these studies found significant high level of LP product such as malondialdehyde [25, 26, 95, 96]. Through this research, Nour Eldin et al. 2014 shows that malondialdehyde concentration in blood as LP biomarker was elevated in T2DM compared to healthy control group, and that there is an associa-

Diabetes mellitus and hypertension are interrelated diseases, and hypertension is about twice as frequent in individuals with diabetes as in those without diabetes. Breath ammonia concentrations in subjects with diabetes can be a consequence of oxidative stress after ROS attack on proteins, accelerated catabolism of proteins due to insulin deprivation and hepatic glucose production [28]. According to Erejuwa, 2012, ROS are involved in insulin signaling, and goes to development of insulin resistance [97]. Therefore, oxidative stress increased insulin resistance and increase the free radical's formation in diabetes, which leads to damage in proteins and lipids. Hyperglycemia generates an increased level of free radicals which can lead to dysfunctions [98, 99] and increases oxidative damage which cause in development of health complications in diabetes, complications associated with inflammation

Some of the subjects with T2DM involved in this study, present complications such as hypertension and/or inflammatory syndrome. T2DM is an inflammatory disease, and inflammation is caused by insulin resistance correlated with obesity, or

compared to those who have no health complications.

*Mean values of HbA1C in n healthy subjects and T2DM.*

tion between hyperglycemia and oxidative stress [27].

[100] or vascular disorders [100, 101].

by hyperglycemia and hyperlipidemia.

#### **Table 1.**

*Mean values for subjects involved with standard deviations (SD).*

*Organic Volatile Compounds Used in Type 2 Diabetes DOI: http://dx.doi.org/10.5772/intechopen.94752*

#### **Figure 5.**

*Type 2 Diabetes - From Pathophysiology to Cyber Systems*

For the subjects involved in this study, among the exhaled breath analyses, were determined blood analyses such as blood glucose levels and glycated hemoglobin HbA1C. **Table 1** shows the values of HbA1c and glucose blood tests, as well as the

Among subjects with T2DM, 7 have hypertension and inflammatory syndrome. In those subjects averages of all analyzes, both blood tests and the breathing are

values of the respiration results obtained with the CO2LPAS system.

**158**

**Table 1.**

**Figure 4.**

**Figure 3.**

**Parameters Healthy** 

Blood glucose (mg dL−1)

**subjects**

*Mean breath ammonia concentrations in healthy subjects and T2DM.*

*Mean breath ethylene concentrations in healthy subjects and T2DM.*

*Mean values for subjects involved with standard deviations (SD).*

**T2DM T2DM with hypertension and** 

83.94 ± 9.2 200.17 ± 44.01 200.17 ± 44.01

HbA1C [%] 4.79 ± 0.52 7.88 ± 0.66 8.21 ± 1.1 *C*C2H4 [ppb] 24 ± 3.35 238 ± 92 246 ± 59 *C*NH3 [ppm] 1.296 ± 0.18 3.96 ± 0.85 5.16 ± 1.12

**inflammatory syndrome**

*Mean values of HbA1C in n healthy subjects and T2DM.*

higher in subjects with T2DM who have hypertension and inflammatory syndrome compared to those who have no health complications.

Our results show a high difference in the HbA1c mean value of healthy group and the and the mean values obtained in subjects with T2DM. These differences are presented in **Figure 5**. In diabetes, impaired detoxification of free radicals and this degree of damage can be seen in HbA1c, which is a biomarker of oxidative stress.

#### **6. Discussions**

Damage to proteins and lipids due to oxidation has been implicated in the pathogenesis of type diabetes. Previous studies on oxidative stress are based on invasive blood samples analysis, and these studies found significant high level of LP product such as malondialdehyde [25, 26, 95, 96]. Through this research, Nour Eldin et al. 2014 shows that malondialdehyde concentration in blood as LP biomarker was elevated in T2DM compared to healthy control group, and that there is an association between hyperglycemia and oxidative stress [27].

Diabetes mellitus and hypertension are interrelated diseases, and hypertension is about twice as frequent in individuals with diabetes as in those without diabetes. Breath ammonia concentrations in subjects with diabetes can be a consequence of oxidative stress after ROS attack on proteins, accelerated catabolism of proteins due to insulin deprivation and hepatic glucose production [28]. According to Erejuwa, 2012, ROS are involved in insulin signaling, and goes to development of insulin resistance [97]. Therefore, oxidative stress increased insulin resistance and increase the free radical's formation in diabetes, which leads to damage in proteins and lipids. Hyperglycemia generates an increased level of free radicals which can lead to dysfunctions [98, 99] and increases oxidative damage which cause in development of health complications in diabetes, complications associated with inflammation [100] or vascular disorders [100, 101].

Some of the subjects with T2DM involved in this study, present complications such as hypertension and/or inflammatory syndrome. T2DM is an inflammatory disease, and inflammation is caused by insulin resistance correlated with obesity, or by hyperglycemia and hyperlipidemia.

Breath ethylene concentrations in subjects with T2DM was found in higher level compared with healthy subjects. Breath ethylene is considered a marker of oxidative stress, being an end-product of LP, caused by the attack of free radicals on polyunsaturated fatty acids. The damage related to free radical's action increase and a direct measurement of the damage can be achieved by quantitative determination of ethylene concentrations.

Ammonia is a biomarker of protein metabolism, and at subjects with T2DM, insulin deprivation is associated with an increase in amino acids and by an accelerated protein catabolism. Moreover, we found that breath ethylene and ammonia concentrations are higher in T2DM subjects that present hypertension and/or inflammatory syndrome than in those without complications. It is known that T2DM lead to complications like kidney failure, heart disease, cerebrovascular disease, but there is a lack of information of ammonia level in subjects with T2DM and the relationship between ammonia level and diabetes complications.

In subjects with T2DM by measuring the percentage of HbA1c, clinicians are able to get an overall picture of the average blood sugar levels have been for the past 2–3 months. Through our measurements, the diabetics present a high level of glycated hemoglobin HbA1c. An increased level of HbA1c reflect a poor metabolic control of the patients with diabetes in uncontrolled T2DM [102–106].

The relation between level of ammonia and ethylene in the exhaled breath and T2DM could be explained by the inadequate insulin control with disease progression by development of complications such as oxidative stress, inflammatory syndrome, and hypertension. The studies show a relation between hyperglycemia, oxidative stress and inflammation coexist in pathological processes but also that hyperglycemia and free radicals increase the oxidative stress which will then activate the inflammatory processes [97, 107].

Future studies are needed to understand the relationship between them and the importance of breath ammonia and ethylene biomarkers.

#### **7. Conclusions**

Real-time breath ethylene and ammonia monitoring in subjects with type-2 diabetes using a CO2LPAS system was realized.

This paper has presented accurate measurement of breath ethylene and ammonia concentrations and the results obtained in comparison with the blood samples analysis have demonstrated the suitability of the experimental PA system for trace gas detection.

This study shows a high level of oxidative stress in people with diabetes through a high level of glycated hemoglobin HbA1c, and high concentrations of ethylene and ammonia in respiration.

Our study suggests that sensitive, noninvasive, real-time analysis of oxidative stress, using ethylene and ammonia as breath biomarkers, distinguishes healthy subjects from those with type 2 diabetes and controlled by uncontrolled diabetes.

The breath analysis may also bring opportunities in molecular monitoring for other research fields by using an LPAS system.

Despite these advances, there is a continuing need for miniaturized devices, in addition to a precise and easy to use instrument, which should provide a quick response, preferably in real time.

**161**

**Author details**

Magurele, Romania

Mioara Petrus\*, Cristina Popa and Ana-Maria Bratu

provided the original work is properly cited.

\*Address all correspondence to: mioara.petrus@inflpr.ro

National Institute for Laser, Plasma and Radiation Physics, Laser Department,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Organic Volatile Compounds Used in Type 2 Diabetes DOI: http://dx.doi.org/10.5772/intechopen.94752*

This work was supported by Romanian Ministry of Education and Research, under Romanian National Nucleu Program LAPLAS VI – contract n. 16 N/2019.

**Funding**

Further research is therefore required to expand the applicability of breath analysis in clinical diagnosis.

*Organic Volatile Compounds Used in Type 2 Diabetes DOI: http://dx.doi.org/10.5772/intechopen.94752*
